Successful Adoptive Immunotherapy Research Articles

CAR-Modified Vγ9Vδ2 T Cells Propagated Using a Novel Bisphosphonate Prodrug for Allogeneic Adoptive Immunotherapy.

The benefits of CAR-T therapy could be expanded to the treatment of solid tumors through the use of derived autologous αβ T cell, but clinical trials of CAR-T therapy for patients with solid tumors have so far been disappointing. CAR-T therapy also faces hurdles due to the time and cost intensive preparation of CAR-T cell products derived from patients as such CAR-T cells are often poor in quality and low in quantity. These inadequacies may be mitigated through the use of third-party donor derived CAR-T cell products which have a potent anti-tumor function but a constrained GVHD property. Vγ9Vδ2 TCR have been shown to exhibit potent antitumor activity but not alloreactivity. Therefore, in this study, CAR-T cells were prepared from Vγ9Vδ2 T (CAR-γδ T) cells which were expanded by using a novel prodrug PTA. CAR-γδ T cells suppressed tumor growth in an antigen specific manner but only during a limited time window. Provision of GITR co-stimulation enhanced anti-tumor function of CAR-γδ T cells. Our present results indicate that, while further optimization of CAR-γδ T cells is necessary, the present results demonstrate that Vγ9Vδ2 T cells are potential source of 'off-the-shelf' CAR-T cell products for successful allogeneic adoptive immunotherapy.

The NOTCH-FOXM1 Axis Plays a Key Role in Mitochondrial Biogenesis in the Induction of Human Stem Cell Memory-like CAR-T Cells.

Recent studies have shown that stem cell memory T (TSCM) cell-like properties are important for successful adoptive immunotherapy by the chimeric antigen receptor-engineered-T (CAR-T) cells. We previously reported that both human and murine-activated T cells are converted into stem cell memory-like T (iTSCM) cells by coculture with stromal OP9 cells expressing the NOTCH ligand. However, the mechanism of NOTCH-mediated iTSCM reprogramming remains to be elucidated. Here, we report that the NOTCH/OP9 system efficiently converted conventional human CAR-T cells into TSCM-like CAR-T, "CAR-iTSCM" cells, and that mitochondrial metabolic reprogramming played a key role in this conversion. NOTCH signaling promoted mitochondrial biogenesis and fatty acid synthesis during iTSCM formation, which are essential for the properties of iTSCM cells. Forkhead box M1 (FOXM1) was identified as a downstream target of NOTCH, which was responsible for these metabolic changes and the subsequent iTSCM differentiation. Like NOTCH-induced CAR-iTSCM cells, FOXM1-induced CAR-iTSCM cells possessed superior antitumor potential compared with conventional CAR-T cells. We propose that NOTCH- or FOXM1-driven CAR-iTSCM formation is an effective strategy for improving cancer immunotherapy. SIGNIFICANCE: Manipulation of signaling and metabolic pathways important for directing production of stem cell memory-like T cells may enable development of improved CAR-T cells.

Impact of various culture conditions on ex vivo expansion of polyclonal T cells for adoptive immunotherapy.

Currently, adoptive immunotherapy is considered as one of the leading treatments in cancer. Successful adoptive immunotherapy depends on producing large numbers of desired T cells ex vivo for infusion. This requires an effective protocol for maximum functional T-cell expansion while keeping the time and costs to a minimum. Current T-cell expansion protocols are diverse in their methodology, and a universal protocol of expansion is wanting. Also, new findings regarding T-cell biology, signaling, and activation have reshaped the strategies of T-cell propagation over the years, introducing new ways to expand T cells. Here, we reviewed different conditions for blood-derived polyclonal T-cell expansion so as to elucidate the influential factors of T-cell expansion and their efficacy.

Reinforce the antitumor activity of CD8+ T cells via glutamine restriction.

The antitumor activity of activated CD8+ T cells in the tumor microenvironment seems to be limited due to their being metabolically unfit. This metabolic unfitness is closely associated with T‐cell exhaustion and impairment of memory formation, which are barriers to successful antitumor adoptive immunotherapy. We therefore assessed the role of glutamine metabolism in the antitumor activity of CD8+ T cells using a tumor‐inoculated mouse model. The adoptive transfer of tumor‐specific CD8+ T cells cultured under glutamine‐restricted (dGln) conditions or CD8+ T cells treated with specific inhibitors of glutamine metabolism efficiently eliminated tumors and led to better survival of tumor‐inoculated mice than with cells cultured under control (Ctrl) conditions. The decreased expression of PD‐1 and increased Ki67 positivity among tumor‐infiltrating CD8+ T cells cultured under dGln conditions suggested that the inhibition of glutamine metabolism prevents CD8+ T‐cell exhaustion in vivo. Furthermore, the transferred CD8+ T cells cultured under dGln conditions expanded more efficiently against secondary OVA stimulation than did CD8+ T cells under Ctrl conditions. We found that the expression of a pro‐survival factor and memory T cell‐related transcription factors was significantly higher in CD8+ T cells cultured under dGln conditions than in those cultured under Ctrl conditions. Given these findings, our study uncovered an important role of glutamine metabolism in the antitumor activity of CD8+ T cells. The novel adoptive transfer of tumor‐specific CD8+ T cells cultured in glutamine‐restricted conditions may be a promising approach to improve the efficacy of cell‐based adoptive immunotherapy.

302 - Ex vivo programming of T cell trafficking and retention within tumors by TGF-β

Adoptive immunotherapy (AI) is a promising approach that has proved effective across different type of tumors including melanoma and Epstein-Barr virus lymphomas. However, despite recent successes, many patients still fail to respond. One of the major challenges for AI is to be able to generate antigen-specific T cells that are capable to effectively traffic and be retained into the tumor. Thus, the development of strategies that could potentially increase infiltration of T cells into neoplastic microenvironments is critical for the success of AI. Transforming Growth Factor beta (TGF-β) is a pleiotropic cytokine mostly known for its immunosuppressive properties. Paradoxically, it has been recently shown that the abundance of TGF-β in the tumor microenvironment may promote T-lymphocyte recruitment and antitumor functions within epithelial cancers. This may occur through the induction of the integrin CD103 expression that promotes effector T-cell adhesion to tumor cells and also triggers intracellular signaling events that provide co-stimulatory signals. Thus, we hypothesise that TGF-β exposure during ex vivo T cell activation for AI may confer a tumor infiltration/retention phenotype to effector T cell within tumors in vivo and hence improve AI efficacy. Using OT-I TcR transgenic model, we found that exogenous TGF-β favors the expression of the integrin CD103 and the chemokine receptor CXCR3 in ex-vivo stimulated OT1-T cells in the presence of the cognate antigen. We also found that TGF-β-treated T cells exhibited strong migration to recombinant CXCL9, CXCL10 and CXCL11, the three CXCR3 ligands. Furthermore, using in vivo competitive homing assays, we found that TGF-β improves T-cell trafficking in Lewis lung carcinoma expressing OVA peptide (LLC-OVA) tumors. Finally, adoptive transfer of TGF-β-treated T cells into LLC-OVA-bearing mice slightly but significantly delayed tumor growth which correlated with higher intratumoral T cell infiltration and maintenance of CD103 expression in vivo. All together, these data suggest that ex-vivo stimulation of antigen-specific T cells with TGF-β may enhance localization within tumor thereby enhancing the anti-tumor response and improving the therapeutic effectiveness of AI.

Constitutive Signaling from an Engineered IL7 Receptor Promotes Durable Tumor Elimination by Tumor-Redirected T Cells.

Successful adoptive T-cell immunotherapy of solid tumors will require improved expansion and cytotoxicity of tumor-directed T cells within tumors. Providing recombinant or transgenic cytokines may produce the desired benefits but is associated with significant toxicities, constraining clinical use. To circumvent this limitation, we constructed a constitutively signaling cytokine receptor, C7R, which potently triggers the IL7 signaling axis but is unresponsive to extracellular cytokine. This strategy augments modified T-cell function following antigen exposure, but avoids stimulating bystander lymphocytes. Coexpressing the C7R with a tumor-directed chimeric antigen receptor (CAR) increased T-cell proliferation, survival, and antitumor activity during repeated exposure to tumor cells, without T-cell dysfunction or autonomous T-cell growth. Furthermore, C7R-coexpressing CAR T cells were active against metastatic neuroblastoma and orthotopic glioblastoma xenograft models even at cell doses that had been ineffective without C7R support. C7R may thus be able to enhance antigen-specific T-cell therapies against cancer.Significance: The constitutively signaling C7R system developed here delivers potent IL7 stimulation to CAR T cells, increasing their persistence and antitumor activity against multiple preclinical tumor models, supporting its clinical development. Cancer Discov; 7(11); 1238-47. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 1201.

4-1BB-Enhanced Expansion of CD8+ TIL from Triple-Negative Breast Cancer Unveils Mutation-Specific CD8+ T Cells.

Triple-negative breast cancer (TNBC) highly infiltrated with CD8+ tumor-infiltrating lymphocytes (TIL) has been associated with improved prognosis. This observation led us to hypothesize that CD8+ TIL could be utilized in autologous adoptive cell therapy for TNBC, although this concept has proven to be challenging, given the difficulty in expanding CD8+ TILs in solid cancers other than in melanoma. To overcome this obstacle, we used an agonistic antibody (urelumab) to a TNFR family member, 4-1BB/CD137, which is expressed by recently activated CD8+ T cells. This approach was first utilized in melanoma and, in this study, led to advantageous growth of TILs for the majority of TNBC tumors tested. The agonistic antibody was only added in the initial setting of the culture and yet favored the propagation of CD8+ TILs from TNBC tumors. These expanded CD8+ TILs were capable of cytotoxic functions and were successfully utilized to demonstrate the presence of immunogenic mutations in autologous TNBC tumor tissue without recognition of the wild-type counterpart. Our findings open the way for a successful adoptive immunotherapy for TNBC. Cancer Immunol Res; 5(6); 439-45. ©2017 AACR.

Dual signals of TCR and Notch promote antigen-specific T cell development from pluripotent stem cells (P4370)

Abstract The pivot in conducting a successful adoptive T-cell immunotherapy for cancer is the obtaining of a large number of naïve phenotyped, stem-cell like tumor antigen specific T cells. Previously we have reported that both conventional and antigen-specific T cells could be generated from induced pluripotent stem (iPS) cells by different methods. Here, we observed that dual signals of TCR and Notch played a significant role in antigen-specific T lineage differentiation from pluripotent stem cells. We used a murine MHC-I restricted, tyrosinase-related protein 2 (TRP2) specific TCR and Notch ligands Delta-like (DLL) 1 and 4 as a model system for inducing iPS cells to differentiate into melanoma-specific CD8+ T cells. Murine iPS cells were retrovirally transduced with TRP2-specific TCR (iPS-TRP2), and then co-cultured on OP9 bone marrow stromal cells overexpressing both DLL1 and DLL4 ligands (OP9-DL1/DL4) in the presence of Flt3L and IL-7. This approach has a better effect in terms of directing an in vitro iPS-TRP2 differentiation towards TRP2-specific CTLs. In addition, adoptive transfer of in vitro developed iPS-TRP2-derived precursor T cells into C57BL/6J mice bearing xenografted B16 melanoma significantly increased anti-tumor immunosurveillance and protected animal from tumor challenge. These data suggest that signals from TCR and Notch can cooperatively promote antigen-specific T cell development from pluripotent stem cells.

Host conditioning approach and choice of target epitope determine the success of adoptive T cell immunotherapy against oncogene-induced established tumors (P2068)

Abstract Host conditioning regimens can dramatically enhance adoptive T cell-based therapies for some cancers. We investigated the unique and combined contributions of whole body irradiation (WBI) and agonist anti-CD40 conditioning to successful adoptive immunotherapy in a mouse model of autochthonous cancer. Line Rip1-Tag4 mice develop pancreatic islet cell tumors due to expression of the SV40 large T antigen oncogene from the rat insulin promoter. Using transfer of T cell receptor transgenic CD8+ T cells specific for two unique determinants in T antigen (designated TCR-I or TCR-IV), we demonstrate that successful control of established tumors in these mice is both regimen- and epitope-dependent. WBI conditioning alone minimally enhanced T cell accumulation and persistence, while anti-CD40 alone transiently enhanced the subdominant TCR-I but not the immunodominant TCR-IV response. Combining anti-CD40 with WBI conditioning enhanced the magnitude and duration of both T cell responses, but only TCR-I cells accumulated and persisted at high levels in the pancreas. Mice that received combined conditioning plus TCR-I transfer had an increased life span, and showed histological signs of anti-tumor immunity. Lack of therapeutic impact by TCR-IV T cells was associated with development of an unusual differentiation phenotype following antigen recognition in vivo. The results indicate that target selection and conditioning regimen dramatically influence the efficacy of adoptive T cell therapy.

A novel system of artificial antigen-presenting cells efficiently stimulates Flu peptide-specific cytotoxic T cells in vitro

Therapeutic numbers of antigen-specific cytotoxic T lymphocytes (CTLs) are key effectors in successful adoptive immunotherapy. However, efficient and reproducible methods to meet the qualification remain poor. To address this issue, we designed the artificial antigen-presenting cell (aAPC) system based on poly(lactic-co-glycolic acid) (PLGA). A modified emulsion method was used for the preparation of PLGA particles encapsulating interleukin-2 (IL-2). Biotinylated molecular ligands for recognition and co-stimulation of T cells were attached to the particle surface through the binding of avidin–biotin. These formed the aAPC system. The function of aAPCs in the proliferation of specific CTLs against human Flu antigen was detected by enzyme-linked immunospot assay (ELISPOT) and MTT staining methods. Finally, we successfully prepared this suitable aAPC system. The results show that IL-2 is released from aAPCs in a sustained manner over 30 days. This dramatically improves the stimulatory capacity of this system as compared to the effect of exogenous addition of cytokine. In addition, our aAPCs promote the proliferation of Flu antigen-specific CTLs more effectively than the autologous cellular APCs. Here, this aAPC platform is proved to be suitable for expansion of human antigen-specific T cells.

Requisite considerations for successful adoptive immunotherapy with engineered T-lymphocytes using tumor antigen-specific T-cell receptor gene transfer

Introduction: Although engineered T-cell-based antitumor immunotherapy using tumor-antigen-specific T-cell receptor (TCR) gene transfer is undoubtedly a promising strategy, a number of studies have revealed that it has several drawbacks.Areas covered: This review covers selected articles detailing recent progress in this field, not only for solid tumors, but also for leukemias. In terms of achieving uniform therapeutic quality of TCR gene-modified T cells as an ‘off-the-shelf’ product, the authors abstract and discuss the requisite conditions for successful outcome, including: i) the optimal target choice reflecting the specificity of the introduced TCR, ii) the quality and quantity of expressed TCRs in gene-modified T cells, and additional genetic modification reflecting enhanced antitumor functionality, and iii) ‘on-’ and ‘off-target’ adverse events caused by the quality of the introduced TCRs and other adverse events related to genetic modification itself. Readers will be able to readily abstract recent advances in TCR gene-transferred T-cell therapy, centering notably on efforts to obtain uniformity in the therapeutic functionality of engineered T cellsExpert opinion: Harmonizing the functionality and target specificity of TCR will allow the establishment of clinically useful adoptive immunotherapy in the near future.

CMV-Reactive γ/δ T Cells Are Equivalent to Their α/β Counterparts In the Lysis of CMV-Infected Targets – Potential for Post Transplant Adoptive Immunotherapy.

AbstractAbstract 1277Reactivation of cytomegalovirus (CMV) post allogeneic stem cell transplantation (SCT) can be prevented and treated by adoptive immunotherapy with donor-derived alpha/beta CMV-reactive T cells (CMV-T). We have reported that VD1+ gamma/delta T cells arising post allogeneic SCT are also CMV-reactive and that these cells can be identified in and isolated from CMV-seronegative donors (Knight et al 2010). The relative ability of VD1 gamma/delta cells to lyse CMV-infected targets compared to CMV-reactive alpha/beta T cells has not been determined.Here we have isolated gamma/delta T cells (VDelta 1) from normal, healthy HLA-A2+, CMV-seropositive donors and tested them freshly isolated and after expansion on allogeneic feeder cells (as described in Knight et al 2010) for their ability to lyse MRC5 fibroblasts infected with CMV strain VHLE in vitro. In parallel we isolated HLA-A2 restricted CMV-reactive alpha/beta T cells using a CMV-specific pentameric HLA reagent (ProImmune Ltd), rested overnight to ensure reagent release and tested for lysis of the same CMV-infected targets.MRC5 fibroblasts were cultured in MEM/10%FCS and infected with VHLE (kind gift from Dr Sinzger, Tubingen, Germany) at an MOI of 1 for three days. Infected cells were recovered by trypsinisation and mixed with effector cells at 5:1 and 10:1 E:T ratios in duplicate in a four hour flow cytometric killing assay. Dead fibroblasts were identified by failure to exclude ToPro Iodide. Control cultures were established with uninfected MRC5 and “percent specific lysis” calculated by subtraction of the degree of lysis of uninfected cells from that of the infected targets at each E:T ratio.Lysis of non-infected MRC5 by both alpha/beta CMV-T cells and VD1 gamma/delta T cells was low (0.7%-13.2%) and was not significantly different between the two cell subsets. In contrast alpha/beta CMV T were highly lytic of CMV-infected targets (mean 33.85, sd 1.48) as were VD1 gamma/delta T cells (mean 26.3% sd 3.53) at 5:1 E:T. There was no significant difference between the specific lysis mediated by alpha/beta CMV-T compared to matched-pair VD1 gamma/delta T cells (p>0.05); nor was there any significant difference in CMV-specific lysis by freshly isolated versus ex-vivo cultured VD1 gamma/delta T cells from these donors. The specific lysis mediated by the VD1 gamma/delta T cells from these CMV seropositive donors was comparable to that we previously reported from CMV seronegative donors although slightly higher.Since clinical trials of alpha/beta CMV-T have shown their protective effect as adoptive immunotherapy post transplant, the fact that we show comparable lytic function by VD1 gamma/delta T cells suggests that these cells may also be clinically useful since they can be readily isolated without functional selection and are known not to mediate GvHD even in the haploidentical transplant setting. More importantly, since the lytic function of VD1 gamma/delta T cells from CMV seropositive donors is comparable to that of VD1 gamma/delta T cells from seronegative individuals we are encouraged that these cells could be used for successful adoptive immunotherapy of CMV in the “CMV+ve receipient:CMV-ve donor” transplant setting. Disclosures:No relevant conflicts of interest to declare.

The biological characteristics of dendritic cells derived in vitro from myelogeneous leukemia cells and healthy donor cells

Successful adoptive immunotherapy for leukemia depends on the generation of T cells that can specifically react with malignant cells. Dendritic cells (DCs) are important antigen-presenting cells in the development of antileukemia T-cell responses. Mononuclear cells (MNC) were isolated from peripheral blood or bone marrow of patients with chronic myelogenous leukemia (CML), and acute myelogenous leukemia (AML). After incubation with granulocyte-macrophage colony-stimulating factor, interleukin (IL)-4, and tumor necrosis factor-alpha, MNC developed morphological characteristics of DCs in vitro, which were confirmed by phenotypic assay. Fluorescence in situ hybridization demonstrated the presence of fusion gene in the nuclei of representative CML or AML-M3 samples, indicating that the cells were leukemic in origin. IL-12 levels were significantly higher in AML-DCs and CML-DCs prestimulated with phorbol 12-myristate 13-acetate than in the corresponding leukemic cells, but were lower than that of healthy donors. These cells were potent stimulators of lymphocyte proliferation in specific in vitro assays for DC function. However, the stimulatory abilities of allogeneic T cells in a mixed lymphocyte reaction were impaired compared with those of mature DCs derived from healthy donors, although T-cell stimulatory effects were significantly increased in these differentiated leukemia-DCs. These results suggest that functional DCs may be derived from leukemic (AML, CML) blasts in a significant number of patients and may be capable of inducing leukemia-specific immune responses with potentially clinically beneficial effects.

CD137-guided isolation and expansion of antigen-specific CD8 cells for potential use in adoptive immunotherapy

The efficient isolation and ex vivo expansion of antigen-specific T cells are crucial for successful adoptive immunotherapy against uncontrollable infections and cancers. Several methods have been reported for this purpose, for example, employing MHC-multimeric complexes, interferon-gamma secretion, and antibodies specific for molecules expressed on T-cell surfaces, including CD25, CD69, CD107a, CD137, and CD154. Of the latter, CD137 has been shown to be one of the most promising targets since it is only expressed on CD8(+) T cells early after encountering antigen, while being almost undetectable on resting cells. However, detailed comparisons between CD137-based and other methods have not yet been conducted. In this study, we therefore compared three approaches (with CD137, CD107a, and tetramers) using HLA-A24-restricted CMV pp65 and EBV BRLF1 epitopes as model antigens. We found that the CD137-based isolation of antigen-stimulated CD8(+) T cells was comparable to tetramer-based sorting in terms of purity and superior to the other two methods in terms of subsequent cell expansion. The method was less applicable to CD4(+) T cells since their CD137 upregulation is not sufficiently high. Collectively, this approach is most likely to be optimal among the methods tested for the isolation and expansion of antigen-specific CD8(+) cells.

Modeling the CD8+ T Effector to Memory Transition in Adoptive T-Cell Antitumor Immunotherapy

Adoptive T-cell therapy with CD8(+) CTLs is often characterized by poor persistence of the transferred T cells and limited effector responses. Improved persistence and therapeutic efficacy have been noted when antigen-activated CD8(+) T cells express properties of memory cells. The current study was undertaken to more precisely characterize the development of memory-like CD8(+) T cells from short-term CTLs in vitro and upon transfer in vivo, including their antitumor activity. Ovalbumin (OVA)-specific OT-I CTLs acquired phenotypic and functional properties of memory cells 2 to 3 days later either by lowering the concentration of antigen to a level that does not support primary responses and providing a survival signal through transgenic Bcl-2 in vitro or simply by transferring early day 3 CTLs to antigen-free lymphoid-replete mice. In lymphoid-replete mice, established OVA-expressing E.G7 tumor was rejected by short-term CTLs that simultaneously acquired memory-like properties in secondary lymphoid tissues, where tumor antigen level remained low. Collectively, these data indicate that CTLs readily converted to memory-like cells upon lowering antigen to a concentration that selectively supports memory responses and suggest that such conversion predicts successful adoptive immunotherapy.

Failure of low-dose recombinant human IL-2 to support the survival of virus-specific CTL clones infused into severe combined immunodeficient foals: Lack of correlation between in vitro activity and in vivo efficacy

Although CTL are important for control of lentiviruses, including equine infectious anemia virus (EIAV), it is not known if CTL can limit lentiviral replication in the absence of CD4 help and neutralizing antibody. Adoptive transfer of EIAV-specific CTL clones into severe combined immunodeficient (SCID) foals could resolve this issue, but it is not known whether exogenous IL-2 administration is sufficient to support the engraftment and proliferation of CTL clones infused into immunodeficient horses. To address this question we adoptively transferred EIAV Rev-specific CTL clones into four EIAV-challenged SCID foals, concurrent with low-dose aldesleukin (180,000 U/m 2), a modified recombinant human IL-2 (rhuIL-2) product. The dose was calculated based on the specific activity on equine PBMC in vitro, and resulted in plasma concentrations considered sufficient to saturate high affinity IL-2 receptors in humans. Despite specific activity on equine PBMC that was equivalent to recombinant equine IL-2 and another form of rhuIL-2, aldesleukin did not support the engraftment and expansion of infused CTL clones, and control of viral load and clinical disease did not occur. It was concluded that survival of Rev-specific CTL clones infused into EIAV-challenged SCID foals was not enhanced by aldesleukin at the doses used in this study, and that in vitro specific activity did not correlate with in vivo efficacy. Successful adoptive immunotherapy with CTL clones in immunodeficient horses will likely require higher doses of rhuIL-2, co-infusion of CD4+ T lymphocytes, or administration of equine IL-2.

Peptide‐β2‐microglobulin‐major histocompatibility complex expressing cells are potent antigen‐presenting cells that can generate specific T cells

Adoptive T-cell therapy represents a promising therapeutic approach for the treatment of cancer. Successful adoptive immunotherapy depends on the ex vivo priming and expansion of antigen-specific T cells. However, the in vitro generation of adequate numbers of functional antigen-specific T cell remains a major obstacle. It is important to develop efficient and reproducible methods to generate high numbers of antigen-specific T cells for adoptive T-cell transfer. We have developed a new artificial antigen-presenting cell (aAPC) by transfection of major histocompatibility (MHC) class I negative Daudi cells with a peptide-beta2-microglobulin-MHC fusion construct (single-chain aAPC) ensuring presentation of the peptide-MHC complex of interest. Using this artificial antigen-presenting cell, we could generate up to 9.2 x 10(8) antigen-specific cytotoxic CD8(+) T cells from 10 ml blood. In vitro generated T cells lysed endogenously presented antigens. Direct comparison of the single-chain aAPC with autologous monocyte-derived dendritic cells demonstrated that these cells were equally efficient in stimulation of T cells. Finally, we were able to generate antigen-specific T cell lines from perpheral blood mononuclear cells of patients receiving cytotoxic chemotherapy. The use of single-chain aAPC represent a promising option for the generation of antigen-specific CD8(+) T cells, which could be used for adoptive T-cell therapy.

Adoptive Immunotherapy against Experimental Visceral Leishmaniasis with CD8+T Cells Requires the Presence of Cognate Antigen

CD8+ T cells have a protective role in experimental visceral leishmaniasis. However, the observation that inflammatory cytokines induce bystander activation of CD8+ T cells questions the need for antigen-dependent effector function. Here, we demonstrate that successful adoptive immunotherapy with CD8+ T cells is strictly dependent upon the presence of cognate antigen.

T-cell reconstitution and expansion after hematopoietic stem cell transplantation: ‘T’ it up!

Adoptive immunotherapy is the isolation and infusion of antigen-specific or nonspecific lymphocytes. Adoptive therapy with T cells may have a role in replacing, repairing, or enhancing immune function damaged by cytotoxic therapies, and rapid lymphocyte recovery may improve outcome after autologous and allogeneic stem cell transplantation (SCT). Recently, a plethora of information on the basic mechanisms of T-cell biology and regulation of cellular immune responses has emerged, permitting the development of new forms of adoptive cell therapy. Efficient ex vivo culture method for T-cell subsets affords the possibility of adoptive transfer of T cells engineered with enhanced capacity for central memory, effector cytotoxicity, Th1, Th2, veto cell, and T regulatory functions. Studies show that homeostatic T-cell proliferation is important for effective adoptive immunotherapy and pretreatment with chemotherapy may enhance the effects of infused T cells. Replicative senescence, in part due to telomere erosion, likely limits successful adoptive immunotherapy, though it may be possible to maintain T-cell pools by enforced expression of telomerase. Clinical trials now demonstrate that it is possible to enhance immune reconstitution after SCT with cytokines or infusions of ex vivo costimulated expanded T cells. These data all support the premise that adoptive therapy can accelerate reconstitution of cellular immunity with enhanced antitumor effects following SCT.

Success of Adoptive Immunotherapy Followed by Stem Cell Transplantation in the Management of Epstein Barr Virus (EBV)-Driven Primary Central Nervous System Lymphoma (PCNSL) in Immunodeficient Child.

EBV-driven PCNSL is a rare form of B-cell non-Hodgkin's lymphoma (NHL) seen in immuno-compromised (particularly AIDs) patients. The prognosis is gloomy and most patients die of their disease as radiotherapy and chemotherapy are usually ineffective in inducing durable remission. We report successful treatment of an 8 years old girl who presented with multifocal EBV-driven B-cell PCNSL with adoptive immunotherapy. Due to rarity of the disease particularly in children, she was investigated for and found to have an underlying primary immunodeficiency associated with defective immunity to herpes viruses. Her underlying immune defect did not fit with any of the currently well characterized congenital immune deficiency syndromes, but as also demonstrated in one of her two brothers, it seemed to have been inherited in an autosomal recessive fashion. Both were persistently lymphopenic (0.3–1.1 X 109/L), with proportionate reduction of T, B, and NK cells. Immunoglobulin classes were normal, as were specific antibody responses to tetanus. Mitogen responses to PHA, serum IgG2 levels and post-vaccination responses to Hib and Pneumococcus were low in both children. Antibody response to EBV was abnormal in both children with development of IgG antibodies to the capsular antigen (VCA) but not the nuclear antigen (EBNA). She was initially treated with conventional chemotherapy (intrathecal as well as systemic high dose Cytarabine and Methrotrexate), Rituximab, anti-virals and immunoglobulins. Neurological deterioration gradually progressed despite four weeks of such treatment. She eventually became bed ridden with quadreparesis and significant bulbar palsy requiring intubation for airway protection. In view of conventional treatment failure, immunotherapy with ex-vivo expanded EBV-specific cytotoxic T-lymphocytes (CTL) from a partially HLA matched donor was initiated. These were given weekly for seven consecutive weeks. There were no untoward effects. She made a remarkable neurological recovery within few weeks and became fully mobile in four months when she received non-myeloablative stem cell transplantation. Post transplant peripheral EBV reactivation (presenting as infectious mononucleosis-like disease without neurological manifestations) was promptly controlled with Rituximab and further doses of allogeneic EBV-CTLs. The child is now well five months post transplantation with evidence of immune reconstitution and remains in complete remission. GVHD prophylaxis has now been totally withdrawn. This is the first report of the successful use of EBV-specific cytotoxic T-lymphocytes in the treatment of primary CNS lymphoma in an immunocompromised patient. It offers new hope in a condition, which until now has been associated with a bleak outlook. The clinical response to immunotherapy is a clear demonstration that although the migration of resting blood lymphocytes across the inter-endothelial tight junctions forming the blood-brain-barrier may be largely restricted in physiological conditions, the migration of activated T lymphocytes into the brain in this clinical setting is unimpeded.